1. Field of the Invention
The present invention relates to treatment of neoplastic disease and more particularly to determining severity of prostate cancer in afflicted patients.
2. Description of Related Art
The ability to monitor neoplastic disease status is an important tool in cancer therapy. In addition to improving prognostication, knowledge of the disease status allows an attending physician to select the most appropriate course of therapy. For example, patients with a high likelihood of relapse can be treated aggressively with powerful systemic chemotherapy and/or radiation therapy. Where there is a lesser likelihood of relapse, less aggressive therapies can be chosen. Since severe patient distress can be caused by more aggressive therapy regimens, it is desirable to determine which patients require such aggressive therapies.
Prostate cancer disease is responsible for nearly 3% of all deaths in men over the age of 55 years. It is likely that more than 300,000 new cases of prostate cancer will be diagnosed in American men this year. Prostate cancer has variable clinical outcome and recent studies indicating the potential benefits of withholding therapy in older men with limited disease and the potential to predict inoperable cancer in men with aggressive tumors has prompted the search for new prognostic markers that could be applied to the initial guided prostate needle biopsy and prove successful in selecting therapy and predicting disease outcome.
The identification of new prognostic markers in prostate cancer would allow urologists to stratify their patients into groups that could receive significantly different therapies. Tumor grade and DNA ploidy have been generally accepted as significant predictors of outcome for the disease (see e.g., Ross et al., Cancer, 74:2811-18(1994)), but a clearly established prognostic panel capable of defining therapy selection has not emerged to date.
Fluorescence in-situ hybridization (FISH) has recently been employed in detection of chromosomal aneusomies and gene copy numbers in both solid tumors and hematopoietic malignancies. See, e.g., Wolman SR., Pathology Annual, Appelton and Lang, Stanford, Conn., pp.227-244 (1995). Using chromosome specific probe, FISH was found to be more sensitive than flow cytometry for the detection of aneuploidy in prostate cancer. Visacorpi et al., Am J Pathol, 145:624-630 (1994). High grade prostate cancer has been linked to chromosomal aneusomy by FISH and chromosome 8 aneusomy has been associated with increased tumor stage. Brown et al., J Urol, 152:1157-1162 (1994). FISH detected aneusomy in prostate cancer has been associated with recurrent and progressive disease. See Lifson et al., Anal Quant Cytol Histol, 17:93-99 (1995); Koivisto et al., Am J. Pathol, 147:16-8-14 (1995); Lieber M M., J Cell Biochem (suppl), 19:246-248 (1994); Bandyk et al., Genes Chrom Cancer, 9:19-27 (1994); Zitzelsberger et al., J Pathol, 172:325-335 (1994); Alcaraz et al., Cancer Res, 54: 3998-4002 (1994). Studies have revealed varying abnormalities associated with disease progression including increased copy number of chromosome X (Koivisto et al., supra) and aneusomies of chromosome 7 and/or 8 (Lieber M M., supra; Bandyk et al., supra; Zitzelsberger et al., supra; Alcaraz et al., supra). FISH based techniques have also been utilized recently to demonstrate potential candidate tumor suppressor genes that may prove of significance in prostate cancer. Ziao et al., Am J Pathol, 147:896-904 (1995); Cher, J Urol, 153:249-254 (1995).
The HER-2/neu (c-erbB2) gene is localized to chromosome 17p and encodes a transmembrane tyrosine kinase growth factor receptor with substantial homology to the epidermal growth factor receptor. HER-2/neu expression in breast cancer has generally been accepted as a predictor of disease outcome with HER-2/neu gene amplification by southern analysis and corresponding overexpression of HER-2/neu protein (p185.sub.neu) by western blotting or immunohistochemistry (IHC) predicting early disease relapse in lymph node negative and lymph node positive patients. See Battifora et al., Modern Pathol, (1991) 4:466-474; Press et al., Cancer Res, (1993)53:4960-4970; Seshadri et al., Clin Oncol, (1993)11:1936-1942; Descotes et al., Anticancer Res, (1993) 13:119-124; Muss et al., N Engl J Med, (1994) 330:1260-1266; Tetu et al., Cancer, (1994) 73:2359-2365; Marks et al., Annal Surg, (1994) 219:332-341. Recently, amplification of the HER-2/neu gene or overexpression of the HER-2/neu protein have been clinically utilized to identify patients likely to be refractory to less intense cytotoxic chemotherapy in breast cancer. Muss et al., supra. Moreover, clinical trials featuring patients with HER-2/neu amplified tumors and therapeutic use of an administered antibody to HER-2/neu protein have shown promise for the treatment of refractory metastic ovarian and breast cancer. See Baselga et al., J Clin Oncol, 14(3):737-44 (1996); Peitras et al., Oncogene, 9(7): 1829-1838 (1994).
In prostate cancer, a consensus as to the predictive value of HER-2/neu gene amplification and p185.sub.neu. protein expression has not been reached. The majority of published prognostic studies of HER-2/neu status in prostate cancer have utilized immunohistochemical techniques featuring a variety of antibodies with differing sensitivities and specificities particularly when utilized in archival specimens. See, e.g., Visacorpi et al., Modern Pathol, (1992) 5:643-648; Ibrihlm et al., Surg Oncol, (1992) 1:151-155; Ross et al., Cancer, (1993)72:3020-3028; Sadasivan et al., J Urol, (1993) 150:126-131; Kuhn et al., J Urol, (1993) 150:1427-1433; Melon et al., J Urol, (1992) 147:496-499. Molecular based studies of the HER-2/neu gene in prostate cancer have been limited to two published reports from one research group which reported an absence of gene amplification by Southern analysis in a small number of prostate cancer specimens, i.e., Latil et al., Int J Cancer, (1994) 59:637-638; Fournier et al., Urol Res, (1995) 22:343-347. In one report using the MAB-1 antibody, no staining could be identified on archival fixed tissue specimens. Visacorpi et al., Modern Path., supra. In another study, immunoreactivity for HER-2/neu oncoprotein was more intense in prostatic hyperplasia and prostatic intraepithelial neoplasia than in adenocarcinoma. Ibrihlm et al., supra. Several previously published immunohistochemical studies of HER-2/neu in prostate cancer have failed to link expression with disease outcome. In one study using the paB-1 antibody on formalin-fixed paraffin-embedded archival material, HER-2/neu oncoprotein expression was identified in one of clinically localized prostate cancers, but did not appear to be a significant prognostic marker. See Kuhn et al., supra. A significant decrease of EGF receptor and increase immunodetection for HER-2/neu protein was identified in prostate cancer but the findings did not correlate with tumor stage or grade. See Melon et al., supra. Finally, in a more recent study of prostate cancer and benign prostatic hyperplasia using the AB-3 antibody on archival tissues, p185.sub.neu immunostaining did not correlate with Gleason grade and a trend toward an inverse relationship was presented. See Gu et al., Cancer Letters, (1996) 99:185-189.
Several immunohistochemical studies of HER-2/neu protein expression in prostate cancer have correlated with other prognostic variables and suggested correlation with disease outcome. In one study using a immunoalkaline phosphatase procedure and the 9G6 antibody, HER/2-neu protein expression was found in 16 of 100 (16%) of prostate cancer specimens and protein expression correlated with high tumor grade and aneuploid DNA content. See Ross et al., supra. In another study utilizing the TA-1 antibody, overexpression of HER-2/neu protein was found to be an indicator of poor prognosis in prostate cancer and correlated with high histologic tumor grade, disease state and DNA aneuploidy. See Sadasivan et al., supra. In a study featuring analysis of a group of potential prognostic markers, HER-2/neu antigenicity was found to be a predictor of prostate cancer progression on univariate analysis and also significantly contributed to further stratification into higher risk of recurrence groups for patient subpopulations initially featuring the usually more favorable low Gleason score tumor grades. See Veltri et al., J Cell Biochem Suppl, (1994) 19:249-258.
Unfortunately, studies of HER-2/neu expression by IHC are subject to considerable technical variations. Given that most specimens are formalin-fixed, paraffin-embedded archival material, false negative staining may occur due to antigen loss. Fixation and processing protocols significantly affect the reactivity of the antigenic determinants detected by HER/2-neu antibodies such as MAB-1 and pAB-60. Ware et al., Hum Pathol, (1991) 22:254-258. Different antibodies may produce either cytoplasmic or membranous staining, be ineffective when certain fixatives are used or be impacted by temperature of the IHC reaction. Ware et al., supra. Antigen retrieval techniques featuring either enzymatic digestion or microwave irradiation contribute additional potential variables that may affect staining levels. Potential sources of error in IHC studies of HER-2/neu oncogene expression in archival breast cancer tissue samples have recently been reported. See Press et al., Cancer Res, (1994) 54:2771-2777. Substantial variation in sensitivity and specificity of commercially available HER-2/neu antibodies to detect gene amplification confirmed by Southern blotting was observed with antibodies such as the pAB-1 featuring 65% sensitivity and the 9G6, 47% sensitivity. Press et al., supra. Fixation and embedding methods similarly affect the results of IHC for HER-2/neu protein in gastric cancer. See Chiu et al., J Clin Pathol, (1994) 47:816-822. Staining interpretation problems and interobserver variability especially concerning cytoplasmic immunoreactivity for HER-2/neu protein have also been reported. See Kay et al., J Clin Pathol, (1994) 47:816-822. The present invention overcomes the above described problems associated with such variation in the immunohistochemical demonstration of HER-2/neu protein in archival tissue specimens.